PROJECT SUMMARY As the second most-common reportable infectious disease in the U.S., gonorrhea is caused by bacterial pathogen Neisseria gonorrhoeae (Ngo) and its treatment has been complicated by the dramatic rise in antimicrobial drug resistance. New treatment options for eliminating this pathogen with novel modes of action are urgently needed. Due to their essential role in maintaining protein homeostasis, 70- kD heat shock proteins (Hsp70s), ubiquitous molecular chaperones, play an important role in bacterial pathogen survival and growth in hosts including Ngo. Hence, Hsp70s represent a promising target for the design of novel and efficient treatments for gonorrhea. However, only a few small molecule inhibitors have been discovered for Hsp70s due to a limited understanding of their chaperone activity. Hsp70s have two nucleotide-bound states: ATP and ADP. For both states, polypeptide substrate binding is crucial for the chaperone activity. The substrate binding mode for the ADP-bound state is well-established: a single strand of polypeptide substrate in extended conformation binds to a narrow substrate binding pocket. In contrast, the substrate binding mode for the ATP-bound state remains a mystery. Our recent crystals structures of Hsp70s in the ATP-bound state revealed a novel conformation of the polypeptide substrate- binding site. This new conformation suggests a novel binding mode for peptide substrates. We hypothesize that Hsp70s in the ATP-bound state bind partially folded polypeptide substrates instead of fully unfolded single-stranded polypeptide. We have designed a partially folded substrate and showed that it has high affinity for the ATP-bound state. More importantly, this partially folded substrate is about 100?1000 times more efficient in inhibiting Hsp70 chaperone activity than previously known single- stranded peptides. Thus, targeting this novel substrate binding mode provides a new direction in designing potent and specific inhibitors for Hsp70s in Ngo infection. The overall objective of this proposal is to design specific and potent inhibitors for Hsp70s by targeting this newly discovered open SBD conformational state and its putative substrate binding modes, and test their effect in inhibiting Ngo. Accordingly, we propose two specific aims: 1) Design inhibitors for Hsp70s that target the open SBD conformational state. We have developed a computational model for partially folded substrates based on our crystal structures of Hsp70s in the ATP-bound state. This model provides an important foundation for computational screening and design of inhibitors that target this novel binding mode. 2) To evaluate the designed inhibitors, we will characterize the biochemical, biophysical and structural properties of their interactions with Hsp70s, and test their potential in inhibiting growth of a number of clinical isolate multi- drug resistant Ngo. Rational design of inhibitors that specifically target Hsp70s as proposed in this application could potentially yield novel and efficient antimicrobial therapeutics against gonorrhea.